Gene therapy is an area of current interest which involves the introduction of genetic material into a cell to facilitate expression of a deficient protein. There are currently five major methods by which this is accomplished, namely: (i) calcium phosphate precipitation, (ii) DEAE-dextran complexes, (iii) electroporation, (iv) cationic lipid complexes and (v) reconstituted viruses or virosomes (see Chang, et al., Focus 10:88 (1988)). Cationic lipid complexes are presently the most effective generally used means of effecting transfection.
A number of different formulations incorporating cationic lipids are commercially available, namely (i) LIPOFECT.RTM. (which uses 1,2-dioleyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTMA, see Eppstein, et al., U.S. Pat. No. 4,897,355); LIPOFECTAMINE.RTM. (which uses DOSPA, see Hawley-Nelson, et al., Focus 15(3):73 (1993)); and LIPOFECTACE.RTM. (which uses N,N-distearyl-N,N-dimethyl-ammonium bromide, or DDAB, see Rose, U.S. Pat. No. 5,279,833). Others have reported alternative cationic lipids that work in essentially the same manner but with different efficiencies, for example 1,2-dioleoyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTAP, see Stomatatos, et al., Biochemistry 27:3917-3925 (1988)); glycerol based lipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990); lipopolyamines (see, Behr, et al., U.S. Pat. No. 5,171,678) and cholesterol based lipids (see Epand, et al., WO 93/105162, and U.S. Pat. No. 5,283,185).
Others have noted that DOTMA and related compounds are significantly more active in transfection assays than their saturated analogues (see, Felgner, et al., W091/16024). However, both DOTMA and DOSPA based formulations, despite being the most efficient of the cationic lipids in effecting transfection, are prohibitively expensive. DDAB on the other hand is inexpensive and readily available from chemical suppliers but is less effective than DOTMA in most cell lines. Another disadvantage of the current lipid systems is that they are not appropriate for intravenous injection.
An examination of the relationship between the chemical structure of the carrier vehicle and its efficiency of transfection has indicated that the characteristics which provide for effective transfection would make a carrier unstable in circulation (see, Ballas, et al., Biochim. Biophys. Acta 939:8-18 (1988)). Additionally, degradation either outside or inside the target cell remains a problem (see, Duzghines, Subcellular Biochemistry 11:195-286 (1985)). Others who have attempted to encapsulate DNA (Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. Pat. No. 4,515,736) made no efforts to ensure a safe, injectable formulation, or arrived at inefficient loading (Legendre, Pharm. Res. 9:1235-1242 (1992)).
Ideally, a delivery vehicle for a nucleic acid or plasmid will have the following characteristics: a) small enough and long lived enough to distribute from local injection sites when given intravenously, b) capable of carrying a large amount of DNA per particle to enable transfection of all sizes of genes and reduce the volume of injection, c) homogenous, d) reproducible, e) protective of DNA from extracellular degradation and f) capable of transfecting target cells in such a way that the DNA is not digested intracellularly.
The present invention provides such compositions and methods for their preparation and use.